Parathyroid hormone antagonists

ABSTRACT

The present invention relates to the use of peptide hormone analogues as inhibitors of their respective naturally occurring peptide hormone. The structure of the peptide hormone analogues is exemplified by parathyroid hormone wherein Gly 12  is substituted by D-Trp, L-Trp, L- or D- alpha- or beta-naphthylalanine, or D- or L- alpha-MeTrp.

RELATED APPLICATIONS

This application is a continuation in part of U.S. Ser. No. 191,512,filed May 9, 1988.

BACKGROUND OF THE INVENTION

This invention relates to the use of peptide hormone analogues forinhibiting the naturally occurring hormone peptide in vivo and in vitro.These peptide hormone analogues when administered to a vertebrate, suchas mammals, block the endrocrine activity of the peptide hormone orother analogous molecules. These peptide hormone analogues are alsouseful in vitro in combination with a bioassay for the naturallyoccurring hormone. The peptide hormone analogues are useful in treatingvarious diseases caused by hormone excess and in treating hormone tumorswhich secrete hormone-like factors that mimic the hormone actions. Oneexample of this invention relates to the synthesis of parathyroidhormone analogues useful for inhibiting the action of parathyroidhormone both in vivo and in vitro.

Analysis of the relation of structure to hormonal function has providedimportant insights into the mechanism of action of peptide hormones.Each type of peptide hormone has an affinity for specific receptors towhich it binds. Upon binding, the peptide hormone acts either directlyor causes a change in the intracellular concentration of a secondmessenger molecule such as cyclic AMP, cyclic GMP, inositol or calciumions. These second messenger molecules, in turn, cause changes in themetabolism or physiology of the cell. These changes in cell metabolismor physiology are directly or indirectly dependent upon the binding ofthe peptide hormone to its specific cell surface receptor. Therefore, ifthe cell surface receptor is blocked then the hormone effect is alsoblocked.

Peptide hormone analogues have long been known as a method through whichthe biochemistry of hormones can be studied and evaluated.Endocrinologists have long desired a method for producing a class ofpeptide hormone analogues which would allow the blocking of specifichormone receptors without activating a change in the second messengermolecules, thereby avoiding the hormone induced metabolic changes.

Rosenblatt et al., U.S. Pat. No. 4,423,037 and the publications referredto therein describe the structure of certain peptide hormone analoguesand their binding to cell receptors. In particular, these publicationsdescribe the properties of parathyroid hormone analogues and theirphysiological properties.

Scientific efforts over a period of many years have sought to understandthe interaction between peptide hormones and the cell surface receptorspecific for each peptide hormone. One of the peptide hormones,parathyroid hormone, has been studied by using analogues of parathyroidhormone (PTH). One objective of these studies has been to understand thebinding of the peptide hormone to the cell surface receptor such that ananalogue could be constructed which would bind with the same or greateraffinity than the naturally occurring hormone. This would enable thepeptide hormone analogue of parathyroid hormone to be used to block theeffect of the naturally occurring parathyroid hormone. One of the majorproblems encountered in this search for a clinically andpharmacologically effective parathyroid hormone analogue was the problemof agonist activity. Agonist activity is the property of the peptidehormone analogue to itself stimulate the change in second messengerswhich brings about the physiological change associated with thenaturally occurring hormone. Therefore, the problem was to createhormone analogues which would bind with high affinity to the appropriatehormone cell surface receptor but not stimulate a change in the secondmessenger concentration, that is, not act as hormone itself. Theseanalogues could then be used in treating hormone related diseases.

The secondary structure analyses of PTH using Chou Fasman parameters(Chou, P. Y. et al., Biophys, J. 26: 367-373 (1979) does not give aclear indication of a beta-turn or a helix in PTH at the region aroundposition 12. The present invention accomplishes a substitution of aminoacids in this region in order to stabilize helix-type or turn-typesecondary structural conformations. It is, therefore, a primary objectof the present invention to stabilize the bioactive conformation of PTHin order to enhance the biological activity of PTH analogues.

Another object of the present invention is to provide novel PTHanalogues. Another object of the present invention is to provide amethod of inhibiting the action of PTH through the administration ofnovel PTH analoges. Still another object of the invention is to providePTH analogues wherein amino acid modifications result in binding to allthe surface receptor without activating the second messenger molecule.The above and other objects are accomplished by the present invention inthe manner more fully described below.

SUMMARY OF THE INVENTION

The present invention provides a peptide which comprises PTH(3-34)NH₂,[Tyr³⁴ ]PTH (3-34)NH₂, [Nle⁸,18,Tyr³⁴ ]PTH(3-34)NH₂ ; PTH(4-34)NH₂,[Tyr³⁴ ]PTH(4-34)NH₂, [Nle⁸,18,Tyr³⁴ ]PTH(4-34)NH₂ ; PTH(5-34)NH₂,[Tyr³⁴ ]PTH (5-34)NH₂, [Nle⁸,18,Tyr³⁴ ]PTH(5-34)NH₂ ; PTH(6-34)NH₂,[Tyr³⁴ ]PTH(6-34)NH₂, [Nle⁸,18,Tyr³⁴ ]PTH(6-34)NH₂ ; PTH(7-34)NH₂,[Tyr³⁴ ]PTH(7-34)NH₂, [Nle⁸,18,Tyr³⁴ ]PTH(7-34)NH₂ wherein Gly¹² issubstituted by D-Trp, L-Trp, L- or D- alpha- or betanaphthylalanine, orD- or L- alpha- MeTrp. The PTH can be human parathyroid hormone (hPTH),bovine parathyroid hormone (bPTH) or rat parathyroid hormone (rPTH).

Some representative examples of the peptide analogues of the presentinvention, are as follows: [D-Trp¹² ]hPTH(7-34)NH₂, [D-Trp¹²,Tyr³⁴ ]hPTH(7-34)NH₂, [D-Trp¹²,Nle⁸,18,Tyr³⁴ ]hPTH (7-34)NH₂ ; [L-Trp¹²]hPTH(7-34)NH₂, [L-Trp¹²,Nle⁸,18,Tyr³ ]hPTH(7-34)NH₂, [L-Tyr¹²,Tyr³⁴]hPTH(7-34)NH₂, [D-Trp¹² ]bPTH(7-34)NH₂, [D-Trp¹², Tyr³⁴ ]bPTH(7-34)NH₂,[D-Trp¹²,Nle⁸,18, Tyr³⁴ ]bPTH(7-34)NH₂ ; [L-Trp¹² ]bPTH(7-34)NH₂,[L-Trp¹², Tyr³⁴ ]bPTH(7-34)NH₂,[L-Trp¹²,Nle⁸,18,Tyr³⁴ ]bPTH(7-34)NH₂ ;[D-Trp¹² ]rPTH(7-34)NH₂, [D-Trp¹²,Tyr³⁴ ]rPTH(7-34)NH₂, [D-Trp¹²,Nle⁸,21, Tyr³⁴ ]rPHT(7-34)NH₂ , [L-Trp¹² ]rPTH(7-34)NH₂,[L-Trp¹²,Tyr³⁴]rPTH(7-34)NH₂, [L-Trp¹²,Nle⁸,21,Tyr³⁴ ]rPTH(7-34)NH₂. Theserepresentative examples should not be construed as limiting theinvention.

The present invention also provides a method of inhibiting the action ofparathyroid hormone comprising the administration of therapeuticallyeffective amount of a parathyroid hormone analogue described above. Thepresent invention also provides a method of treating osteoporosis orhypercalcemia comprising the administration of a therapeuticallyeffective amount of a parathyroid hormone analogue described above. Amethod of treating hyperparathyroidism comprising the administration ofa therapeutically effective amount of the parathyroid hormone analoguesof this invention is also provided. A method of treatinghyperparathyroidism expressed as a hypercalcemic crisis, renal failureor hypertension is also provided. A method of treating the disease stateproduced by a tumor or other cell overproducing a peptide hormone-likemolecule and method of treating immune diseases wherein the diseasestate comprises inflammation, an allergic response, or hyperactivelymphocytes is also provided by the novel peptide hormone analogues ofthe present invention.

DETAILED DESCRIPTION OF THE INVENTION

Various other objects, features and attendent advantages of the presentinvention will be more fully appreciated as the same becomes betterunderstood from the following detailed description.

Extensive structure and activity studies have now led to the design ofpeptide hormone analogues which have high binding affinity for theirrespective cell surface receptors while not stimulating the productionof second messenger molecules. Examples of such a peptide hormoneanalogues are [D-Trp¹²,Tyr³⁴ ]hPTH(7-34)NH₂ and [D-Trp¹², Tyr³⁴]bPTH(7-34)NH₂ which inhibit PTH in vitro but do not act as an agonists.

Agonist activity is dependent upon the presence of the N-terminal aminoacid sequence. The removal of two to six end terminal amino acidsresults in the loss of most if not all agonist activities. Therefore,the second messenger molecules are not affected by those analogues whichhave the altered amino terminus.

PTH analogues with two to six amino acids removed from the N terminusproduces an inhibitor which still binds with high affinity to thepeptide hormone receptor without causing a change in cyclic AMPconcentration.

The following is the 34-amino acid sequence of bovine parathyroidhormone (bPTH):

    H2N-ALA-VAL-SER-GLU-ILE-GLN-PHE-MET-HIS-ASN-LEU-GLY-LYS-HIS-LEU(15)-SER-SER-MET-GLU-ARG-VAL-GLU-TRP-LEU-ARG-LYS-LYS-LEU-GLN-ASP(30)-VAL-HIS-ASN-PHE-COOH.

The following is the 34-amino acid sequence of human parathyroid hormone(hPTH):

    H2N-SER-VAL-SER-GLU-ILE-GLN-LEU-MET-HIS-ASN(10)-LEU-GLY-LYS-HIS-LEU-ASN-SER-MET-GLU-ARG(20)-VAL-GLU-TRP-LEU-ARG-LYS-LYS-LEU-GLN-ASP(30)-VAL-HIS-ASN-PHE-COOH.

The following is the 34-amino acid sequence of rat parathyroid hormone(rPTH):

    H2N-ALA-VAL-SER-GLU-ILE-GLN-LEU-MET-HIS-ASN(10)-LEU-GLY-LYS-HIS-LEU-ALA-SER-VAL-GLU-ARG(20)-MET-GLN-TRP-LEU-ARG-LYS-LYS-LEU-GLN-ASP(30)-VAL-HIS-ASN-PHE-COOH.

Fragments of peptide hormones containing the region specific for bindingto the cell surface receptor can be used as inhibitors or blockingagents. For parathyroid hormone, the N-terminal 34 amino acids aresufficient to define binding specificity to the parathyroid hormone cellsurface receptor. This receptor specificity is further defined by thefollowing publication herein incorporated by reference: M. Rosenblatt,et al., Endocrinology, 107:2. 545-550, 1980 and S. R. Nussbaum, et al.,Journal of Biological Chemistry, 255:10183, 1980.

The presence of D-amino acids in peptide hormone in place of L-aminoacids results in a peptide resistant to catabolism. However, not allsuch substitutions result in an active peptide hormone. The insertion oftyrosine amide at position 34 in PTH results in a significant increasein the biological activity of the hormone in addition to increasingstability of the peptide. The utilization of D-amino acids in peptidehormone synthesis is described in the following publications hereinincorporated by reference: Coltrera, et al., Biochemistry, 19:4380-4385,1980; Rosenblatt et al., Biochemistry, 20:7246-7250, 1981.

The balance of the description will be divided into two sections.Section I will describe the preparation and structure of inhibitors ofpeptide hormones, Section II will discuss the use of the peptide hormoneinhibitors.

I. Preparation and Structure of Peptide Hormone Inhibitors

The technique of solid-phase peptide synthesis, developed by Merrifield("Solid Phase Peptide Synthesis", Advances in Enzymology, 32:221-296,1969) and G. Barany & R. B. Merrifield "Solid Phase Peptide Synthesis"in The Peptides Volume 2, editors E. Gross & J. Meienhofen (1980) hasbeen successfully employed in the synthesis of peptide hormonesincluding parathyroid hormone. This method is based on the strategy ofhaving the carboxyl terminus of the peptide linked covalently to a solidsupport. The desired peptide sequence is prepared by stepwise couplingof single amino acids to a peptide chain growing from the carboxyltoward the amino terminus. Coupling is typically achieved by activationof the carboxyl group of the amino acid being attached to the resin,which may have other potentially reactive groups blocked. Followingaddition of an amino acid to the growing polypeptide chain, and prior tofurther chain elongation, the α-amino protecting (BOC) group istypically removed. Because each amino acid is coupled by nearly the sameseries of reactions, the need for elaborate strategies in the synthesisis minimized. Solubility is not a major issue during synthesis, becausethe peptide is linked to a solid support. This method is rapid and itcan be utilized by a single worker. It is very convenient for thesynthesis of multiple analogues with amino terminal substitutions,because a single synthesis can be branched in multiple directions nearthe amino terminus, thereby creating many analogues varying only in theamino terminal region.

II. Use of Peptide Hormone Inhibitors

The method of inhibiting the action of peptide hormones comprises theadministration of a therapeutically effective amount of any peptidehormone or analogue of the present invention. These hormone analoguesretain specificity for the cell surface receptor without stimulating aphysiological response. This method of use applies to the entire peptidehormone or its analogue, or to a fragment of the peptide hormonecontaining the receptor binding site.

The use of peptide hormone analogues is exemplified by parathyroidhormone analogues. The parathyroid hormone may be of bovine, human, rat,or any vertebrate origin. The analogue may contain all the amino acidsexcept for the modified N-terminal region or it might comprise theN-terminal 7-34 amino acids. Individual amino acids can be substitutedto improve biological or chemical stability as exemplified in thepresent invention.

The peptide hormone analogues of this invention can be used in vitro tomeasure the concentration of naturally occurring peptide hormone. Thisbioassay procedure is illustrated by a bioassay for parathyroid hormone.The unknown concentration of parathyroid hormone in a solution can bedetermined by measuring the amount of parathyroid hormone analoguerequired to inhibit its binding to the parathyroid hormone cell surfacereceptor. The concentration of PTH analogue required to block the actionof parathyroid hormone is a direct indicator of the parathyroid hormoneconcentration.

Parathyroid hormone analogues can be used to diagnose the etiology of orto treat osteoporosis or hypercalcemia through the administration of atherapeutically effective amount of the parathyroid hormone analogues ofthis invention. Similarly, hyperparathyroidism and other aspects ofhyperparathyroidism, such as a hypercalcemic crisis, renal failure orhypertension can be treated through the administration of theparathyroid hormone analogues of this invention.

Tumors and other aberrant cell growth often produce hormone-likesubstances causing a disease state. The use of peptide hormone analoguesto block stimulation caused by such hormone-like substances can resultin the alleviation of the disease state. Therefore, the peptide hormoneanalogues of the present invention can be administered to treat diseasescaused by aberrant production of hormone-like substances.

Immune diseases such as inflammation, allergic responses and hyperactivelympocytes can be treated through the administration of peptide hormoneanalogues which block the action of peptide hormones, such as PTHanalogues inhibiting the binding of PTH to cells of the immune system.

The peptide hormone analogues of this invention exhibit both oral andparenteral activity and can be formulated in dosage forms for oral,parenteral, intra-nasal, or topical administration. Solid dosage formsfor oral administration include capsules, tablets, pills, powders andgranules. In such solid dosage forms, the active compound is admixedwith at least one inert diluent such as sucrose, lactose or starch. Suchdosage forms can also comprise, as is normal practice, additionalsubstances other than inert diluent. In the case of capsules, tablets,and pills, the dosage forms may also comprise buffering agents. Tabletsand pills can additionally be prepared with an enteric coating.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsion, solutions, suspensions, syrups and elixerscontaining inert diluents commonly used in the pharmaceutical art.Besides inert diluents, such compositions can also include adjuvants,such as wetting agents, emulsifying and suspending agents, andsweetening. Preparations according to this invention for parenteraladministration include sterile aqueous or non-aqueous solutions,suspensions or emulsions. Examples of non-aqueous solvents or vehiclesare propylene glycol, polyethylene glycol, vegetable oils such as oliveoil and injectable organic esters such as ethyloleate.

The dosage of active ingredient in the compositions of this inventionmay be varied; however it is necessary that the amount of the activeingredient shall be such that a suitable dosage form is obtained. Theselected dosage form depends upon the desired therapeutic effect, theroute of administration, and the duration of the treatment.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

EXAMPLE 1 Synthesis and Purification of Peptide Hormone Analogues of PTH

Analogues of parathyroid hormone, were prepared by a modification of thesolid phase method of Merrifield. Syntheses were performed using anApplied Biosystems 430A Synthesizer. 4-Methylbenzhydrylaminehydrochloride resin (polystyrene-1% by divinylbenzene, USB) was employedas the solid support in order to effect the carboxyamide (CONH2)COOH-terminal modification.

The tertiary butyloxycarbonyl (Boc) group was used to protect the alpha-amino group of each amino acid during coupling. Side-functionprotection was afforded as follows: (a) the hydroxyl group of serine wasprotected as the O-benzyl ether (BZL); (b) the hydroxyl group oftryosine as the 0-2,6dichlorobenzyl ether (DCB) orp-bromobenzyloxycarbonyl ester (BrZ); (c) the carboxyl group of glutamicand aspartic acid as the benzyl (BZ) or cyclohexyl ester (Chx); (d) theimidazole nitrogen of histidine by the benzyloxymethyl (BOM) and theguanidine function of arginine was protected by the p-toluene-sulfonyl(TOS) group, and the indole imine by formyl groups (FOR); and (e) thelysine epsilon amino group by 2-chlorobenzylcarbonyl (CLZ). All aminoacids were obtained from Applied Biosystems, Inc., PeninsulaLaboratories, or Bachem Chemicals.

The peptide-resin synthesis were carried out using Applied Biosystems,Inc. specified protocols. Double couplings were carried out for theincorporation of each amino acid. Deprotection times withtrifluoroacetic acid were extended 6 minutes over manufacturerprotocols.

The peptide was cleaved from the copolymer resin with simultaneousremoval of the side-chain protecting groups similar to the 2 step HFcleavage procedure described by Tam, J.A.C.S. 105: 6442-6455 (1983). Inthe first HF step the following ratios of reagents were used: 5%p-cresol, 5% p-thiocresol, 65% dimethyl sulfide and 25% HF. 10 ml ofmixture per gram of peptide-resin was used for 2 hours at 0° C. In thesecond HF step the following ratio of reagents were used: 5% p-cresol,5% p-thiocresol and 90% HF. The cleavage was carried out for 75 min. at0° C. After removal of HF the peptide-resin mixture was washed withanhydrous ether to remove scavenger. The peptide was then extracted with50% acetic acid and water. The washes were combined and chromatographedusing Sephadex G 50F, eluting with 50% HOAc.

After lyophilization, the partially Purified peptide was chromatographedby reverse phase HPLC (Vydac C₄ bonded silica, 15 u particle size, 300 Åpore size, using aqueous acetonitrile gradient containing 0.1% TFA).

EXAMPLE 2 PTH Binding Assay Results

PTH analogues were analysed in a new receptor assay (Goldman et al.,Endocrin. (1988) 123:1468-1475) which modified the assay reported inRosenblatt et al., Endocrin. 107: 545-550 (1980). The binding assay used[Nle⁸,18, ¹²⁵ I-Tyr³⁴ ]bPTH (1-34)NH₂ which was purified by HPLC(Novapak C₁₈, 32-35% CH₃ CN in 0.1% TFA) and was stored as aliquots in25 mM TrisHCl/1% BSA at -70° C. Bovine renal cortical plasma membraneswere incubated with radioligand (25,000 cpm) in the absence or presenceof PTH analogues in a Tris containing buffer (250 μl) for 30 min. at 21°C. Once equilibrium was reached, bound and free radioligand wereseparated by centrifugation. High specific binding (85%) to bovine renalcortical membranes was obtained consistently.

                  TABLE                                                           ______________________________________                                                               Binding                                                Structure              K.sub.I (nM)                                           ______________________________________                                        [D--Trp.sup.12,Tyr.sup.34 ]bPTH(7-34)NH.sub.2                                                        8 ± 1                                               [Nle.sup.8,18,D--Trp.sup.12,Tyr.sup.34 ]bPTH(7-34)NH.sub.2                                           13 ± 3                                              [Tyr.sup.34 ]bPTH(7-34)NH.sub.2                                                                       76 ± 9.0                                           [Nle.sup.8,18,Trp.sup.34 ]bPTH(7-34)NH.sub.2                                                         145 ± 13                                            [Trp.sup.12,Tyr.sup.34 ]bPTH(7-34)NH.sub.2                                                           106 ± 9                                             [Nle.sup.8,18,Trp.sup.12,Tyr.sup.34 ]bPTH(7-34)NH.sub.2                                              51 ± 3                                              ______________________________________                                    

EXAMPLE 3 ROS 17/2.8 ASSAY RESULTS

PTH analogues were analyzed in a rat osteosarcoma cell line (ROS 17/2.8)for their ability to inhibit cyclic AMP stimulation by 1 nM[Nle⁸,18,Tyr³⁴ ]bPTH(1-34)NH₂ by the method described in R. J. Majeskaet al, Endocrinol. 107, 1494 (1980). Briefly ells, preloaded with [³ H]adenine were incubated with the agonist PTH(1-34) in the presence orabsence of the PTH analogue for 5 minutes at 37° C. in the presence ofIBMX (phoaphodiesterase inhibitor). The formation of [³ H] cAMP wasfollowed to determine the effect of the pTH analogue.

    ______________________________________                                                             Adenylate Cyclase                                        Structure            K.sub.I (nM)                                             ______________________________________                                        [D--Trp.sup.12,Tyr.sup.34 ]bPTH(7-34)NH.sub.2                                                      211 ± 116                                             [D--Trp.sup.12,Nle.sup.8,18,Tyr.sup.34 ]bPTH(7-34)NH.sub.2                                         69 ± 17                                               [Tyr.sup.34 ]bPTH(7-34)NH.sub.2                                                                    2720 ± 520                                            ______________________________________                                    

EXAMPLE 4 Bovine Renal Membrace Adenylate Cyclase

PTH analogues were evaluated for their effects on the stimulation ofcyclic AMP production by bovine renal membranes stimulated with 3nM[Nle⁸,18,Tyr³⁴ ]bPTH-(1-34)NH₂. Bovine renal membranes were incubatedwith agonist in the presence or absence of the PTH analogue at 30° C for30 min. Cyclic AMP produced was measured as described (B. L. Brown et.al., Adv. Cyclic Neucleotide Res. 2,24 (1972)).

    ______________________________________                                                            Adenylate Cyclase                                         Structure           K.sub.I (nM)                                              ______________________________________                                        [D--Trp.sup.12,Tyr.sup.34 ]bPTH(7-34)NH.sub.2                                                     69 ± 5                                                 [Nle.sup.8,18,D--Trp.sup.12,Try.sup.34 ]                                                          125 ± 7                                                bPTH(7-34)NH.sub.2                                                            [Tyr.sup.34 ]bPTH(7-34)NH.sub.2                                                                   880 ± 70                                               [Nle.sup.8,18,Tyr.sup.34 ]bPTH(7-34)NH.sub.2                                                      1631 ± 350                                             [Trp.sup.12,Tyr.sup.34 ]bPTH(7-34)NH.sub.2                                                         737 ± 105                                             ______________________________________                                    

EXAMPLE 5 PTH Binding Assay Results

PTH analogs were analyzed for their ability to inhibit I¹²⁵ -labeled PTHbinding to the bone-derived rat osteosarcoma cell line ROS 17/2.8 asdescribed in McKee, R. et al., Endocrinology (1988) 122 (6): 3008-10.The results are as follows:

    ______________________________________                                                             Binding                                                  Structure            K.sub.I (nM)                                             ______________________________________                                        [D--Trp.sup.12,Tyr.sup.34 ]bPTH(7-34)NH.sub.2                                                      123                                                      [Nle.sup.8,18,D--Trp.sup.12,Tyr.sup.34 ]                                                           182 ± 32                                              bPTH(7-34)NH.sub.2                                                            [Nle.sup.8,18,Tyr.sup.34 ]bPTH(7-34)NH.sub.2                                                        964 ± 170                                            [Trp.sup.12,Tyr.sup.34 ]bPTH(7-34)NH.sub.2                                                         397 ± 21                                              [Tyr.sup.34 ]bPTH(7-34)NH.sub.2                                                                    700 ± 90                                              ______________________________________                                    

EXAMPLE 6 PTH-binding Assay Results (Canine Kidney)

PTH analogues were assayed for their ability to inhibit I¹²⁵ -labeledPTH binding to canine renal certical membranes in an assay similar tothat described in Example 2.

    ______________________________________                                                             Binding                                                  Structure            K.sub.I (nM)                                             ______________________________________                                        [Tyr.sup.34 ]bPTH(7-34)NH.sub.2                                                                    45.8 ± 11.3                                           [D--Trp.sup.12,Tyr.sup.34 ]bPTH(7-34)NH.sub.2                                                      0.38 ± 0.14                                           ______________________________________                                    

EXAMPLE 7 PTH-binding Assay Results (Human osteosarcoma cells)

PTH analogues were assayed for their ability to inhibit I¹²⁵ -labeledPTH binding to human osteosarcoma cells (BIO) in a manner similar tothat described in Example 5.

    ______________________________________                                                            Binding                                                   Structure           K.sub.I (nM)                                              ______________________________________                                        [Tyr.sup.34 ]bPTH(7-34)NH.sub.2                                                                   5.5 ± 1.6                                              [D--Trp.sup.12,Tyr.sup.34 ]bPTH(7-34)                                                             3.5 ± 1.6                                              ______________________________________                                    

What is claimed is:
 1. A peptide having the formula PTH(7-34)NH₂,(Tyr³⁴)PTH(7-34)NH₂ or (Nle⁸,18,Tyr³⁴)PTH (7-34)NH₂, wherein Gly¹² issubstituted by D-Trp.
 2. A peptide according to claim 1, wherein PTH isrPTH, bPTH or hPTH.
 3. A peptide according to claim 2 which is [D-Trp¹²]hPTH(7-34)NH₂, [D-Trp¹²,Tyr³⁴ ]hPTH(7-34)NH₂ or [D-Trp¹²,Nle⁸,18,Tyr³⁴]hPTH(7-34NH₂.
 4. A peptide according to claim 2 which is [D-Trp¹²]bPTH(7-34)NH₂, [D-Trp¹²,Tyr³⁴ ]bPTH(7-34)NH₂ or [D-Trp¹²,Nle⁸,18,Tyr³⁴]bPTH(7-34)NH₂.
 5. A peptide according to claim 2 which is [D-Trp¹²]rPTH(7-34)NH₂, [D-Trp¹²,Tyr³⁴ ]rPTH(7-34)NH₂ or [D-Trp¹²,Nle⁸,18,Tyr³⁴]rPTH(7-34)NH₂.
 6. A method of acting upon a PTH receptor whichcomprises administering a hypercalcemia treating effective amount of apeptide of claim 1 to a mammal.
 7. A pharmaceutical composition whichcomprises a hypercalcemia treating effective amount of a peptide ofclaim 1 and a pharmaceutically acceptable carrier.